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Cell Research, (2001); 11(1):37-43
Hydrogen peroxide-induced changes in intracellular pH of guard
cells precede stomatal closure
ZHANG XIAO1,2, FA CAI DONG1, JU N FENG GAO2, CHUN PENG SONG1,*
1
2
Department of Biology, Henan University, Kaifeng 475001, China
College of Life Sciences, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100,
China
ABSTRACT
Epidermal bioassay demonstrated that benzylamine, a membrane-permeable weak base, can mimick
hydrogen peroxide (H2O2) to induce stomatal closure, and butyric acid, a membrane-permeable weak acid, can
partly abolish the H2O2-induced stomatal closure. Confocal pH mapping with the probe 5-(and-6)-carboxy
seminaphthorhodafluor-1-acetoxymethylester (SNARF-1-AM) revealed that H2O2 leads to rapid changes in
cytoplasmic and vacuolar pH in guard cells of Vicia faba L, i. e. alkalinization of cytoplasmic areas occur red
in parallel with a decrease of the vacuolar pH, and that butyric acid pretreatment can abolish alkalinization
of cytoplasmic areas and acidification of vacuolar areas of guard cells challenged with H2O2. These results imply
that the alkalinization of cytoplasm via efflux of cytosol protons into the vacuole in guard cells challenged
with H2O2 is important at an early stage in the signal cascade leading to stomatal closure.
Key words: H2O2, pH, SNARF-1-AM, confocal, Vicia guard cell.
INTRODUCTION
Even under optimal conditions, many metabolic
processes, including chloroplastic, mitochondrial,
and plasma membrane-linked electron transport
systems, produce reactive oxygen species (ROS)
such as the superoxide radical (O2-), hydrogen peroxide (H2O2), and the hydroxyl free radical (OH-)[1,
2]. Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of ROS, resulting in oxidative damage
at the cellular level. Interestingly, ROS appear to
play a crucial role in several metabolic processes of
plants in a beneficial manner[3]. H2O2, in particular,
have been previously implicated as a second messenger in plant cells exposed to environmental
stresses such as chilling[4], heat[5], and pathogens
[6, 7]. The evidences that O2- and H2O2 involved in
*Corresponding author:
E-mail: [email protected]
Tel: 0378-2874021,2865799
Received Nov-9-2000 Reviced Jan-2-2001 Accepted Jan-31-2001
regulation of stomatal movement have been found
[8], and also oxidative stress resulting from exposure to methyl viologen (which generates O 2radicals) or H2O2 has a marked effect on stomatal
aperture[9], and exogenous H2O2 (
10-5M) can
induce stomatal closure and inhibit stomatal opening without any damage to cells[10]. Recent studies using recombinant aequorin techniques have
demonstrated that H2O2 stimulates a transient increase in whole-plant calcium concentration ([Ca2+]
i)in tobacco seedling[11]. In addition, ozone (O3)
has been shown to inhibit stomatal opening by inactivating the inward-rectifying K+ channel in
plasma membrane[12]. Moreover, our previous
work using voltage clamp had demonstrated that
externally applied H2O2 could induce stomatal closing by the inhibition of K+ uptake and evoking K+
release through K+ channels in plasma membrane
of guard cells[13]. It has been widely confirmed
that H2O2 regulates stomatal movement as a stress
H2O2-induced pH changes precede sstomatal closure
signal, yet there remain considerable gaps in our
knowledge and in our attempts to construct a detailed description of the events and underlying signaling chains involved in the process of stomatal
opening and closing.
Intracellular pH changes have long been proposed to function in signaling and transport control in plant cells, such as plant defense responses
[14], tip growth [15], [16], nodulation[17], elicitation of benzophenanthridine alkaloids[18], and
response to hormone activity such as gibberellic acid
[19] and abscisic acid[20]. To date, cytoplasmic alkalinization is a major step in the ABA-triggered
signal cascade in guard cells leading to H+ efflux
and stomatal closure[21], [22], and internal and
external ABA can induce the generation of H2O2 in
guard cells (manuscript to be submitted). However,
up to now, no conclusive evidence has been provided concerning the earliest signals for stomatal
closure by H2O2. Therefore, in the present paper we
report dynamic changes of the intracellular pH distribution observed by confocal pH topography in
guard cells challenged with H2O2.
MATERIALS AND METHODS
Chemicals and reagents
SNARF-1-AM was purchased from Molecular Probes (Eugene,
OR). Nigericin, benzylamine and butyric acid were from Sigma.
Dimethyl sulfoxide (DMSO) was from Amresco. Pluronic F-127
was from Bio-Rad. Unless stated otherwise, other chemicals
were of analytical grade.
Plant material
Broad bean (Vicia faba L.) was grown from seed in pots with
culture soil (nutrient soil/ vermiculite 2:1). The plants were kept
in a greenhouse with a humidity of about 70%, a photon flux
density of 0.20 to 0.30 mmol m-2 s-1 and day/night temperature of
25 2 oC/20 2 oC. There were no stresses on plants during
growing period.
Epidermal strip bioassay
Stomatal bioassay experiments were performed as described
by McAinsh et.al.[10] with slight modifications. Immediately
prior to each experiment, the epidermis was peeled carefully
from the abaxial surface of the youngest and fully expanded
leaves of 4-week-old plants, and cut into pieces of 5-mm length.
To study promotion of stomatal closure by H2O2, freshly prepared abaxial epidermis was first incubated in CO2-free 50 mM
KCl/10 mM Mes, pH 6.15 (Mes-KCl) for 3 h under conditions
promoting stomatal opening (at 22 oC to 25 oC, under a photon
flux density of 0.20 to 0.30 mM m-2 s-1) to open the stomata, and
then transferred to CO2-free Mes-KCl in the presence of H2O2 (10-
38
5M) or benzylamine (10 mM) or butyrate (10 mM) for 2 h under
opening conditions, and stomatal apertures were determined
microscopically at 30-min intervals. Values are the means of
120 measurements SE values.
Loading with SNARF-1-AM
The uncharged SNARF-1-AM can passively diffuse across
the plasma membrane to cytosol, where it is hydrolyzed by cellular esterases to free SNARF-1. It is this form which is fluorescent,
and is consequently the pH probe. The epidermal strips, which
were first incubated for 3 h under conditions promoting stomatal opening, were placed in a small Petri dish containing 1 ml
CO2 -free Mes-KCl buffer (pH 6.15), with 20 M SNARF-1-AM
from a 3 mM stock solution in DMSO, and were incubated for 30
min in the dark at 25-27oC. The fluorescence probe uptake was
further facilitated when 0.05% pluronic F-127 was present. Loading was terminated by rinsing the strips several times in dyefree incubation buffer.
Confocal pH topography
Intracellular pH measurement and pH calibration using
single excitation-dual emission fluorescence ratios were performed according to the method developed by Roos et al[18],
[23]. Briefly, a confocal laser scanning microscope (Bio-Rad
MicroRadiance) was used in dual-channel mode. Wavelength
settings for SNARF-1 were Ex=488nm; Em=580nm (channel
1) and Em > 600 nm (channel 2). Images were obtained with a
Nikon TE300 inverted microscope (objective 40 /0.60 Plan
Fluor). Signals from selected frames were scanned simultaneously in both channels and the intensity ratio (channel 1 :
channel 2) was calculated for each pixel. pH maps were obtained
by color coding these intensity ratios according to self-defined
look-up tables, as supplied by the manufacturer. To enable the
comparison of changes in signal intensity, confocal images were
taken using identical exposure conditions (in manual setup) for
all samples. Experiments were repeated at least three times with
reproducible results, and the selected confocal image was the
representative of about eight pictures.
The measured intensity ratios were converted to pH by means
of a calibration graph. For this purpose, SNARF-preloaded epidermal strips were incubated in Mes buffers, pH 5.5-7.5 (adjusted
with Tris), containing 50 mM KCl, 10 mM Mes, 10 μM butyrate,
10 mM nigericin (a K+/H+ exchanger which results in a equilibration of external and internal pH), for another 20 min, then the
ratio-versus-pH curve were obtained by scanning pH maps of
above cells and averaging to yield fluorescence ratios at defined
pH values (Fig 1). Changes in intracellular pH can be calculated
as a linear relationship between pH and fluorescence intensity
ratios over the pH ranges 5.5-7.5.
RESULTS
Changes in stomatal behavior in response to
H2O2, benzylamine and butyric acid
To determine whether an intracellular pH
change is involved in stomatal closure by H2O2, we
Zhang X et al
measured the effects of weak base (benzylamine)
and acid (butyric acid) on stomatal movement. As
shown in Fig 2 and Fig 3, 10 mM benzylamine,
similar to 10-5M H2O2, induced stomatal closure,
and 10 mM butyric acid partly abolished the H2O2induced stomatal closure. However, treatment with
10 mM butyric acid alone induced a slight stomatal
opening over untreated controls. These indicated
that guard cell alkalinization occurred in response
to H2O2, and cytosolic alkalinization was possibly an
upstream factor of stomatal closure induced by
H2O2.
Fig 1. Calibration of pH-dependent fluorescence of carboxy
SNARF-1. Fluorescence intensity ratios are averaged from
at least 5 values, each representing the mean intensity ratio of an area over the whole guard cell tested.
Fig 2. Effect of benzylamine (10 mM) and H2O2 (10-5 M)
on the stomatal closure.
H2O2-induced changes in intracellular pH of
guard cells
Having established that cytosolic alkalinization
was involved in the regulation of stomatal closure
induced by H2O2 in above epidermal strips bioassay
experiments, we then considered whether external H2O2 induces changes in intracellular pH of
guard cells or not. In this study, subcellular regions
were delineated by using bright-field and pH-sensitive fluorescence ratio analysis to determine which
cellular compartments had undergone changes in
fluorescence intensity. Time-course quantitative
analysis was performed in the cytosol (area 1, 3, 5)
and the vacuole (area 2, 4). As shown in Fig 4, the
mean pHs of the cytoplasmic and vacuolar areas
stayed about 7.0 and 5.9 respectively in guard cell
without treatment (Fig 4 D). After adding 10-5M
H2O2, the mean cytoplasmic pH increased by around
0.3 unit in parallel with the mean vacuolar pH decreasing by around 0.4 unit (Fig 4 H, Fig 4 I). Following H2O2 treatment, an increase in intracellular pH also occurred in the surrounding epidermal
cells (decreasing background fluorescence ratio as
in Fig 4), which is in accordance with the effect of
ABA on stomatal closure[24].
Effects of butyric acid on changes in intracellular pH of guard cells induced by H2O2
The data presented above raise the question of
whether an increase of the cytoplasmic pH alone is
sufficient to produce a signal for the induction of
stomatal closure. By using a 10 min treatment with
Fig 3. M Effect of butyric acid (10 mM) on the promotion
of stomatal closure by H2O2.
39
H2O2-induced pH changes precede sstomatal closure
butyric acid (a membrane-permeable weak acid
that has frequently been used to decrease the intracellular pH of various cell types in experiments
[14], [25]), we were able to evoke an acidification
of the cytoplasm. As shown in Fig 5, butyric acid
(in Mes-KCl buffer with pH adjusted to 6.15) pretreatment lowered significantly the intracellular pH
in both cytoplasmic and vacuolar areas, especially
the latter (compare the pH maps D in Fig 4 and
Fig 5 at zero min before the addition of H2O2). With
the prolongation of H2O2 treatment, the mean cytoplasmic pH stayed around 6.8 and that of vacuolar
pH showed no drop but a slight increase (compare
Fig 5 G with Fig 4 I), both indicating that butyric
treatment can abolish partly the cytosolic alkalin-
ization induced by H2O2.
DISCUSSION
Despite the recognition of H2O2 as a central
signaling molecule in stress and wounding
responses, pathogen defense, and regulation of cell
cycle and cell death, little is known about how the
signal is perceived and transduced in plant cells.
Previous studies have unraveled that H2O2, similar
to ABA, regulates stomatal movement via an increase in [Ca2+]i as a second messenger[10,11].
Intracellular pH regulation is a characteristic and
necessary feature of all living cells. There is increasing evidences that suggest changes in intracellular
pH can act as a second messenger in plants[14,26,
Fig 4. pH distribution of an open stomata of broad
bean guard cells after the addition of 10-5 M H2O2.
Photograph A is the transmission light image. Photographs
B and C are the fluorescence intensity images acquired from
channel 1 (Em 580 nm) and channel 2 (Em>600 nm) respectively at 0 min before the addition of H2O2. Photograph
D is a pH map derived by ratioing the image intensities
(channel 1/channel 2) at 0 min before the addition of H2O2;
circles point to analyzed areas with vacuoles (2, 4) and cytoplasm (1, 3, 5). Photograph E, F, G, H are pH maps derived
by ratioing the image intensities (channel 1/channel 2) at 1
min, 2 min, 3 min and 4 min after the addition of H2O2
respectively. Fig I is the time-course plot of fluorescence
ratios of selected areas in photograph D. The pseudocolor
bar in photograph H represents a pH range from about 5.5
in vacuolar area to 7.5 in cytoplasmic area. Bar=10 μm.
40
Zhang X et al
Fig 5. Effects of 10 min pretreatment with butyric
acid on changes of intracellular pH in guard cells
challenged with 10 -5 M H 2O 2. Prior to experiment,
SNARF-1-loaded epidermis were incubated in Mes-KCl
buffer containing 10 m M butyric acid for 10 min. Photograph A is the transmission light image. Photographs B and
C are the fluorescence image intensities acquired from channel 1 (Em 580 nm) and channel 2 (Em>600 nm) respectively at 0 min before the addition of H2O2. Photograph D is
a pH map derived by ratioing the image intensities (channel
1/channel 2) at 0 min before the addition of H2O2; circles
point to analyzed areas with vacuoles (1, 4) and cytoplasm
(2, 3, 5). Photographs E, F, are pH maps derived by ratioing
the image intensities (channel 1/channel 2) at 1 min, 4 min
after the addition of H2O2 respectively. Fig G is the timecourse plot of fluorescence ratios of selected areas in photograph D. The pseudocolor bar in photograph F represents a
pH range from about 5.5 to 7.5 as in Fig 4, Bar=10 μm.
27]. Unlike intracellular free Ca2+ concentrations,
which can rapidly change by perhaps 100-fold, the
pH inside a cell varies by only fractions of a pH
unit, but even with such small change, it still exerts many physiological roles[15].
The data presented here, for the firsttime as
far as our knowledge, is concerned that intracellular pH changes are involved in H2O2-induced stomatal closure. Epidermal strip bioassay has shown
that the cytoplasmic alkalinization possibly mediated H2O2-induced stomatal closure (Fig 2, Fig 3),
which is similar to results seen in guard cells in
response to ABA[21,24]. To provide further evidence for the specific involvement of cytoplasmic
alkalinization in H2O2 signaling leading to stomatal
closure and to investigate the shifts of intracellular
pH distribution, we used pH-sensitive SNARF-1AM to directly measure the fast intracellular pH
changes. SNARF-1 enters cells in the diacetate form
(SNARF-1-AM), and is then hydrolyzed by cellular
esterases and trapped as SNARF-1, which is a
single-excitation dual-emission fluorescent probe
and its optical properties make it amenable to
ratiometric imaging by confocal laser scanning
microscopy to measure quantitatively intracellular pH changes. Additionally, ratiometric measurements reduce or eliminate variations of several determining factors in measuring fluorescence intensity, including indicator (dye or probe)
concentration, excitation pathlength, excitation
intensity and detection efficiency, and many artifacts are also eliminated including
photobleaching and leakage of the indicator, variable cell thinkness, and nonuniform indicator distribution within cells (due to compartmentalization).
A single cell assay illustrated that H2O2 induces
rapid changes in cytoplasmic and vacuolar pH in
guard cells of Vicia faba, i.e. alkalinization of cytoplasmic areas occurred in parallel with a decrease of the vacuolar pH, and that butyric acid
41
H2O2-induced pH changes precede sstomatal closure
pretreatment partly abolished alkalinization of cytoplasmic areas and acidification of vacuolar areas
of guard cells challenged with H2O2 (Fig 4 and Fig
5), which are coincident with the epidermal bioassay (Fig 2 and Fig 3). These results imply that the
alkalinization of the cytosol may be accomplished
via a H+ efflux from cytosol to vacuole through
vacuolar H+-ATPase. Proton gradient across tonoplast is possible a necessary factor in H2O2-induced
stomatal closure.
In addition, ratiometric fluorescence of the
edges of guard cell challenged with H2O2 is more
significant than that in the same cell with H2O2-free
treatment (Fig 4), and this implies that H2O2 possibly activates plasma membrane H+-ATPase, which
will cause a more acidification of cell wall. In plant
cells, plasma membrane H+-ATPase is primarily
responsible for generating membrane potential
[28,29]. Therefore, changes in membrane potential may cause or may be a reflection of changes in
cytosolic pH that are involved in signaling leading
to stomatal closure. Taken together with other recent data[10,12,13,21,24], we postulate that H2O2induced stomatal closure is probably mediated by
the alkalinization of cytoplasm, intracellular Ca2+
increase and depolarization of plasma membrane
leading to K+ channel changes. However the spatiotemporal relationship between pH and Ca2+ as well
as plasma membrane K+ channel in H2O2 signaling
leading to stomatal closure remains unknown.
ACKNOWLEDGEMENT
This work was supported by National Natural
Science Foundation of China (No. 39870372) and
Henan Natural Science Foundation (No.
994012300).
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